This project will investigate the delivery of an intracellular antioxidant protein to central neurons using nanomedicine-based technologies for the improved treatment of neuro-cardiovascular diseases. Angiotensin II (AngII), the primary effectors peptide of the renin-angiotensin system, plays a critical role in cardiovascular and body fluid homeostasis. Dysregulation of AngII-dependent neural mechanisms and elevated superoxide (O2O-) are implicated in the pathogenesis of brain-related cardiovascular diseases, including hypertension and heart failure. Previous studies have shown that adenoviral-mediated overexpression of the O2O- scavenging enzyme copper/zinc superoxide dismutase (CuZnSOD) attenuates central AngII-dependent cardiovascular dysfunction. However, the therapeutic potential for adenoviral vectors is weakened by toxicity and the inability of adenovirus to target the brain when peripherally administered. Other studies have modified CuZnSOD with polyethylene glycol (PEG) to improve its circulating half-life; however, PEGylation decreases the permeability of proteins across the blood-brain barrier (BBB). To circumvent these issues, the proposed studies will utilize a nanomedicine-based delivery system in which CuZnSOD protein is electrostatically bound to a synthetic poly(ethyleneimine)-polyethylene glycol (PEI-PEG) polymer to form a stable polyion complex, so-called CuZnSOD nanozyme (SOD-nano). This project will test the hypothesis that SOD-nano is transported into central neurons via active endocytosis and that following peripheral administration is taken up by neurons in AngII-sensitive brain regions which lack a BBB. In Specific Aim 1, the mechanism by which SOD-nano enters neurons will be investigated using fluorescently-labeled SOD-nano, inhibitors of clathrin and/or caveolae- mediated endocytosis, and confocal microscopy. In Specific Aim 2, immunohistochemical analysis of brain and other tissues and whole animal in vivo imaging will be conducted to elucidate the biodistribution and potential neurotoxicity of SOD-nano following peripheral (intravenous) administration. In Specific Aim 3, the effect of peripherally administered SOD-nano on the central AngII-induced pressure response will be determined in mice using radiotelemetry to measure in vivo neuro-cardiovascular physiological responses. The proposed studies will provide new insight into how antioxidant proteins (e.g., CuZnSOD) can be modified with nanocarriers to penetrate neurons in the brain. Notably, the impact of these studies extends beyond pathogenesis of brain- related cardiovascular diseases. Identification of nanoformulations to increase brain delivery and neuronal uptake of antioxidant proteins may be applicable to other central nervous system disorders associated with elevated levels of reactive oxygen species, including Alzheimer's disease, Parkinson's disease, stroke, and traumatic brain injury. In addition to the proposed scientific research, this fellowship will support the applicant's participation in nanotechnology-based coursework and career development.